Pneumococcal surface protein A (PspA) is a leading vaccine candidate to protect against Streptococcus pneumoniae (Sp) infections. This grant addresses issues critical to defining the mechanisms of action of PspA and antibody (Ab) to PspA. This information is important to our understanding of the pathogenesis of Sp and to the potential use of PspA as a component of a common-protein vaccine. The proposed experiments constitute some of the last pre-clinical studies needed prior to renewed human trials with PspA. New and existing information about the mechanism of action of both PspA and Ab to PspA will be used to develop and test the relevance of in vitro surrogate assays for in vivo protection mediated by Ab to PspA. Relevant surrogate assays are needed to evaluate immune sera from phase I and II human safety and immunogenicity trials. The information gained will provide a scientific basis for decisions about whether the much more expensive phase III efficacy trials should be conducted. PspA can inhibit C' activation and surface deposition on Sp. Ab to PspA can enhance C' deposition on Sp. Our studies should also reveal the mechanism by which PspA blocks C' activation. In studies that will include transgenic and KO mice, we will evaluate the effects of Ab to PspA on phagocytosis, immune adherence, and blood clearance of Sp. PspA also keeps apolactoferrin from killing Sp by neutralizing lactoferrin and its cationic peptide lactoferricin. Ab to PspA prevents the PspA-lactoferrin interaction and, thus, enhances apolactoferrin's ability to kill Sp. We will use a lactoferrin KO mouse to help determine if these effects have in vivo relevance. We will also determine if PspA can also prevent killing by other cationic peptides. We will use a panel of MAb to different domains and epitopes of PspA and paired pre-and post-immune (anti- PspA) human sera to help us determine which of the our mechanistic assays of protection with Ab to PspA are best able to predict the ability of the same MAb to passively protect mice from infection. The results obtained will allow us to identify both those mechanisms that are critical for in vivo protection and in vitro assays that will best serve as in vitro surrogates of in vivo protection mediated by antibodies to PspA. Finally, the proposal includes an expanded investigation of our observation that immunization with the proline-rich (PR) domain of PspA can elicit protection against infection. This finding may turn out to be extremely important to the use of PspA as a vaccine because of a prior observation that some Ab to the protection-eliciting coiled coil (CC) domain of PspA can react with denatured myosin at room temperature. Although there is no compelling reason to believe that this observation has clinical relevance, it has caused nervousness on the part of for-profit vaccine companies. If, as we expect, immunization with the PR domain is broadly efficacious and cross- protective, the PR domain will not only provide a means of circumventing the theoretical worries about the CC domain but will also be a more effective immunogen. PUBLIC HEALTH RELEVANCE: This application describes pre-clinical studies to determine the virulence mechanisms of Streptococcus pneumoniae protein PspA and the protective action of antibody elicited to it. This information is critical to understand the role of PspA in pathogenesis and to our development herein of in vitro surrogate assays of the in vivo protection mediated by Ab to PspA. These surrogate assays are needed to guide the development of clinical vaccine trials from phase I through phase III. A protein vaccine against S. pneumoniae is important because of the high complexity and cost of the polysaccharide-protein conjugate vaccine and the rapid evolution of pneumococci to evade the capsule-type-specific protection elicited by the conjugate vaccine.